臺灣博碩士論文加值系統

本研究以回應曲面法分別進行綠殭菌（Nomuraea rileyi CCRC 35515）液態與固態發酵，最適化生產菌絲體和分生孢子之探討。在液態發酵方面，以葡萄糖、果糖、蔗糖、麥芽糖與糖蜜為碳源，其中以葡萄糖為最佳；另一方面，在酵母萃出物、neopeptone與玉米浸粉的氮源試驗中，則以玉米浸粉為最佳。於葡萄糖及玉米浸粉培養基中添加綜合蔬果汁（V8 juice）具有促進菌體生長之功效，而搖瓶培養的其它物理因子：培養液起始酸鹼值以pH 6 為最佳，菌體絲乾重亦隨搖瓶轉速上升而提高。以回應曲面法所得之最適化培養基組成為葡萄糖3.2﹪、V8 juice 29﹪及玉米浸粉0.15﹪，可得到最高菌絲體乾重為12.1 g/L。將此最適化培養基條件應用於5 L液態發酵槽試驗中，在1 vvm通氣量條件下，較高的發酵槽攪拌速率可得到較佳的菌絲體產量，且排出氣體的含氧量較低；但是當通氣量為2 vvm時，不同攪拌速率（250、350及450 rpm）對菌絲體生長並無明顯影響，而菌絲體乾重可高達16.4 g/L。
在固態發酵方面，RSM同樣應用在最適化分生孢子的生產，糖蜜、玉米浸粉、酵母粉、魚漿及高梁仁等培養基對綠殭菌生長最佳，且產孢量可達4.79×109 conidia/g-dry weight。在固態發酵量產方面，將平板及太空包之結果轉移至22-L固態發酵槽，以2800 Lumens/m2且光照週期為L/D＝12/12，其產孢量最佳可達6.8×109 conidia/g-dry weight。
固態發酵槽所生產之分生孢子，其對四齡甜菜夜蛾的致死率為56.05±5.43﹪，與SMAY斜面培養基比較其活性並無明顯差異。

The purpose of this research was studying the optima production of mycelia and conidia spore of Nomuraea rileyi by using the response surface method in submerged cultural fermentation and solid-stat fermentation, respectively. In submerged cultural fermentation, the glucose was found to be the best carbon source for the growth of mycelia among glucose, fructose, sucrose, maltose, and molasses. On the other hand, the corn steep powder was the better nitrogen in the selected testing nitrogen source such as yeast extract, peptone, and corn steep powder. The V8 juice as the additive was found the promoting mycelia growth effect in the media with glucose and corn steep powder. Other physical conditions in the shake flask study, the pH6 as initial growth pH and higher shaker rotation speed were also found favor the mycelia growth. The optima growth compositions from the RSM was 3.2% glucose, 29% V8 juice, and 15% corn steep powder resulted 12.1 g-cell dry weight per liter. This result was applied to scale up in the 5-L fermentor. The higher agitation speed of the fermentor has higher cell production and has lower oxygen contain in the output gas with 1 vvm aeration. However, at higher aeration of 2 vvm the speed of mixing was not affected significant at 250 rpm, 350 rpm, and 450 rpm. Finally, the best cell production was found as high as 16.4 g-cell dry weight per liter.
In solid-stat fermentation, the RSM was also applied in the optima spore production. The molasses, corn steep powder, yeast powder, fishmeal, and sorghum were found to be the best medium for the growth and conidia spore production with 4.76×109 conidia/g-dry weight. The scale up study of solid-stat fermentation was carrying out from the plate, plastic bag, and all the way to 22-L bioreactor. In 22-L bioreactor the higher spore production 6.8×109 conidia/g-dry weight was found at light intensity 2800-Lumens/m2 and light period of L/D=12/12.
Against 4-th star larva of Spodoptera exigua the conidia spore from the bioreactor has 56.05±5.43％ mortality. This result was closes to the conidia spore from the SMAY slant.

封面內頁
簽名頁
大葉大學碩士論文全文授權書 iii
中文摘要 iv
英文摘要 vi
誌謝 viii
目錄 ix
圖目錄 xii
表目錄 xv
第一章 緒論 1
第二章 文獻回顧 3
2.1 綠殭菌之背景及特性 3
2.2 化學農藥對綠殭菌抑制作用 5
2.3 綠殭菌對哺乳類動物之安全性 6
2.4 營養需求及環境因子對綠殭菌產孢之影響 7
2.5 農產品廢棄物之應用 8
2.6 固態與液態發酵 9
2.7 回應曲面法 11
2.7.1回應曲面法之原理 11
2.7.2二水準因子設計 13
2.7.3陡升路徑法 14
2.7.4中心混成設計 15
2.7.5回應曲面模式適切性之統計檢驗 16
第三章 實驗材料與方法 17
3.1 試驗設備 17
3.2 試驗材料 17
3.2.1 試驗培養基 18
3.2.2 菌種 18
3.3 試驗方法 19
3.3.1 種菌製備 19
3.3.2分析方法 19
3.3.3 液態發酵最適化培養條件之探討 20
3.3.4 固態發酵最適化培養條件之探討 28
第四章 結果與討論 38
4.1 綠殭菌液態發酵最適化培養條件之探討 38
4.1.1 培養天數及起始酸鹼值對生長之影響 38
4.1.2 搖瓶轉速對生長之影響 38
4.1.3 綜合蔬果汁對生長之影響 41
4.1.4 碳源對生長之影響 41
4.1.5 氮源對生長之影響 46
4.1.6 基本培養液與碳、氮源之交互作用對生長之影響 46
4.1.7 培養基最適組成份之探討 46
4.1.8 液態發酵槽操作因子對生長之影響 60
4.2以固態發酵方式生產綠殭菌分生孢子之探討 67
4.2.1接種濃度對產孢量的影響 67
4.2.2光照週期對產孢量的影響 67
4.2.3維他命添加液對產孢量的影響 72
4.2.4碳源對產孢量的影響 72
4.2.5氮源對產孢量的影響 76
4.2.6培養基質對產孢量的影響 76
4.2.7培養基最適組成份之探討 79
4.2.8 固態發酵槽（系統一）光照強度對產孢量之影響 90
4.2.9固態發酵槽（系統二）量產試驗 90
第五章 結論與展望 97
參考文獻 100
附錄一、米酒酒槽水之主要成分 108
附錄二、回應曲面進行步驟流程圖 109
附錄三、培養基之碳、氮、氫元素分析表 110
附錄四、酵母粉及玉米浸粉之成份表 111
附錄五、22L固態發酵槽示意圖 112
附錄六、220 L 固態發酵槽示意圖 113
圖目錄
頁次
圖 4-1 起始酸鹼值及培養時間對綠殭菌液態培養之影響 39
圖 4-2 培養箱轉速對綠殭菌液態培養之影響 40
圖 4-3 不同濃度酒水對綠殭菌液態培養之影響 42
圖 4-4 不同濃度V8 juice對綠殭菌液態培養之影響（接種孢
子懸浮液） 43
圖 4-5 不同濃度V8 juice對綠殭菌液態培養之影響（接種菌
絲體） 44
圖 4-6 不同碳源對綠殭菌液態培養之影響 45
圖 4-7 不同氮源對綠殭菌液態培養之影響 47
圖 4-8 不同濃度V8 juice添加碳、氮源對綠殭菌液態培養之
影響 48
圖 4-9 玉米浸粉與葡萄糖對綠殭菌菌絲體乾重之回應曲面圖 54
圖 4-10 玉米浸粉與葡萄糖對綠殭菌菌絲體乾重之等高線圖 55
圖 4-11 玉米浸粉與V8 juice對綠殭菌菌絲體乾重之回應曲面圖 56
圖 4-12 玉米浸粉與V8 juice對綠殭菌菌絲體乾重之等高線圖 57
圖 4-13 葡萄糖與V8 juice對綠殭菌菌絲體乾重之回應曲面圖 58
圖 4-14 葡萄糖與V8 juice對綠殭菌菌絲體乾重之回應曲面圖 59
圖 4-15 液態發酵槽通氣量對綠殭菌液態培養之影響 62
圖 4-16 液態發酵槽通氣量對綠殭菌氧氣消耗速率之影響 63
圖 4-17 液態發酵槽攪拌速率對綠殭菌液態培養之影響 64
圖 4-18 液態發酵槽攪拌速率對綠殭菌氧氣消耗速率之影響 65
圖 4-19 液態發酵槽氧氣質傳對綠殭菌液態培養之影響 66
圖4-20、不同攪拌速率對綠殭菌於液態發酵槽培養之影響（非中心混成點） 68
圖 4-21 液態發酵槽氧氣質傳對綠殭菌氧氣消耗速率之影響 69
圖 4-22 接種不同濃度孢子懸浮液對綠殭菌產孢量之影響 70
圖 4-23 不同光照週期對綠殭菌產孢量之影響 71
圖 4-24 不同濃度酒槽水對綠殭菌產孢量之影響 73
圖 4-25 不同濃度V8 juice對綠殭菌產孢量之影響 74
圖 4-26 不同碳源對綠殭菌產孢量之影響 75
圖 4-27 不同氮源對綠殭菌產孢量之影響 77
圖 4-28 不同培養基質及培養基組成份對綠殭菌產孢量之影響 78
圖 4-29 玉米浸粉與糖蜜對綠殭菌產孢量之回應曲面圖 84
圖 4-30 玉米浸粉與糖蜜對綠殭菌產孢量之等高線圖 85
圖 4-31 玉米浸粉與V8 juice對綠殭菌產孢量之回應曲面圖 86
圖 4-32 玉米浸粉與V8 juice對綠殭菌產孢量之等高線圖 87
圖 4-33 V8 juice與糖蜜對綠殭菌產孢量之回應曲面圖 88
圖 4-34 V8 juice與糖蜜對綠殭菌產孢量之等高線圖 89
圖 4-35 固態發酵槽光照強度對綠殭菌產孢量之影響 92
圖 4-36 22L 固態發酵槽以光照強度2800 Lumens/m2 所生產之綠殭菌孢子經熱處理之發芽率 94
圖 4-37 22L 固態發酵槽以光照強度6500 Lux 所生產之綠殭菌孢子經熱處理之發芽率 95
表目錄
頁次
表 3-1 25-1部分因子實驗設計 28
表 3-2 25-1部分因子設計中各自變數的水準與相對濃度 24
表 3-3 陡升路徑實驗設計 25
表 3-4 中心混成實驗設計 26
表 3-5 25-1部分因子實驗設計 27
表 3-6 25-1部分因子設計中各自變數的水準與相對濃度 31
表 3-7 陡升路徑實驗設計 33
表 3-8 中心混成實驗設計 34
表 4-1 25-1部分因子設計及實驗結果 50
表 4-2 根據25-1 部分因子設計實驗結果所進行之陡升路徑 51
表 4-3 中心混成設計及其實驗結果 53
表 4-4 實際實驗數據與回應模式所預估的預測值比較表 61
表 4-5 25-1部分因子設計及實驗結果 80
表 4-6 根據25-1 部分因子設計實驗結果所進行之陡升路徑 81
表 4-7 中心混成設計及其實驗結果 83
表 4-8 實際實驗數據與回應模式所預估的預測值比較表 91
表 4-9 固態發酵槽不同光照強度所生產之分生孢子粉經水浴加
熱處理對綠殭菌感染甜菜夜蛾四齡幼蟲之致死率 93

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